The amino acid sequences of liver microsomal membrane proteins will be determined and used to interpret their role of specific segments of these molecules in the overall function of the molecule and in their interaction with other proteins and lipids in the membrane. I propose to continue the study of the primary structures and active sites of cytochrome P-450, its reductase, and the stearyl-CoA desaturase. The active site and orientation studies will focus on the phenobarbital induced cytochrome P-450, epoxide hydrolase and the cytochrome b-5 reductase. The amino acid sequences of the first two proteins were recently determined in our laboratory, and the primary structure of cytochrome b-5 reductase is also essentially completed. The methodology includes isolation of these proteins from microsomes of normal and induced rabbit/rat livers. Sequencing strategy will employ reverse phase HPLC of peptides generated by chemical and enzymatic cleavages followed by automated sequence analysis. Active site studies will be performed by labeling the proteins with group specific reagents followed by characterization of the labeled peptide(s) and amino acids. The orientation studies will include incorporation of their active forms in synthetic lipid vesicles, followed by application of specific surface probes, and then by isolation and sequence analysis of labeled fragments to establish the exact sequence position of the labeled residues. Such studies should contribute to an understanding of the structure function relationship of membrane-bound proteins. The health-relatedness of this project is that a preponderence of xenobiotics, including chemical carcinogens and drugs as well as such physiological compounds as steroids and prostaglandins are metabolized by the cytochrome P-450's system. Cytochrome b-5 system also functions as an electron donor for the cytochrome P-450, but more importantly, it is electron donor for the stearyl-CoA desaturase which is the key enzyme for synthesis of unsaturated fatty acids and thus may be implicated in hypercholesterolemia, which is a constant finding in athlerosclerosis.